Introduction
Ectomycorrhizal fungi are widespread in forest ecosystems. Major genera include Amanita, Boletus, Pisolithus, Russula, Suillus, and Xanthoconium. These ectomycorrhizal fungi have a symbiotic relationship with tree roots and thus contain root metabolites, such as enzymes, hormones, and bioflavonoids [1]. Kikuchi et al. [2] demonstrated that the flavonoids secreted by Pinus densiflora play roles in basidiospore germination of Suillus bovinus. Ectomycorrhizal fungi cover the root surface and penetrate through epidermal cells into the cortex. During ectomycorrhizal development, the inner tissue structures of ectomycorrhizal fungi, called hartig nets, are formed and exchange major nutrients in cortical cell zone [3] to improve plant growth and development in ecosystems [4, 5].
In this study, we attempted to induce in vitro mycorrhization of four species ectomy-corrhizal fungi, namely, Amanita spissacea NIFoS 2719, Pisolithus arhizus NIFoS 2784, Suillus spraguei NIFoS 2848, and Xanthoconium affine NIFoS 2716 in P. densiflora. Optimal culture media compositions for fungal growth were determined using solid and liquid media. These four species produce epigeous fruit bodies in the wild and are widespread in coniferous or mixed forests. However, artificial mycorrhizal symbiosis of these four species with P. densiflora has not been explored.
Materials and Methods
Fungal and plant materials
All fungal species used in this study were obtained from the National Institute of Forest Science (NIFoS). A. spissacea NIFoS 2719, P. arhizus NIFoS 2784, S. spraguei NIFoS 2848, and X. affine NIFoS 2716 were isolated from fruiting bodies in 2014 (Table 1). Samples were grown on potato dextrose agar (PDA; Difco, Detroit, MI, USA) at 25°C for 60 days and used as inocula. P. densiflora seeds were collected from trees in the National Institute of Forest Science (NIFoS) and stored at 4°C. Seed surfaces were sterilized in 30% H2O2 for 30 min. Seeds were germinated on PDA media at 25°C for one week.
Mycelial growth on media
Mycelial growth on solid media. All species were inoculated on 85 mm petri dishes. For culture on solid media, we examined four culture media, namely, PDA, malt extract agar (MEA; Difco, Detroit, MI, USA), Sabouraud dextrose agar (SDA; Difco, Detroit, MI, USA), and modified Melin-Norkran’s medium [MMN; 10 g of glucose, 0.5 g of KH2PO4, 0.15 g of MgSO4․7H2O, 1.2 mL of FeCl3 (1% sol.), 0.05 g of CaCl2, 0.025 g of NaCl, 3 g of malt extract, 0.25 g of (NH4)2HPO4, and 100 µg of thiamin×HCl in 1 L of water]. Cultures were incubated at 25°C for 60 days.
Mycelial growth on liquid media. The four liquid media, namely, MMN, M0, M1, and M2, were used for this study; MMN medium with 10 g/L glucose as a carbon source instead of sucrose, M0 with no nitrogen source, M1 with ammonium nitrogen [(NH4)Cl] as nitrogen source, and M2 with nitrate nitrogen (KNO3) as nitrogen source.
Mycorrhizal induction
Liquid inocula of fungal species. For each species, a small sample (6 mm × 6 mm) was cut and inoculated in a 500 mL Erlenmeyer flask containing 200 mL of sterile MMN broth. Fungal cultures were incubated at 25°C for 60 days.
Mycorrhizal induction. For mycorrhizal induction, 1 L culture bottles were added with 600 mL of granite soil and 100 mL of PDMP (6 g of potato dextrose broth, 0.75 g of malt extract, and 0.25 g of peptone per liter). Culture bottles were then autoclaved at 100°C and 121°C consecutively for 20 min. Germinated seeds were transferred into sterilized culture bottles and grown for one week. Afterwards, each seedling was injected with liquid inoculum. Inoculated seedlings were grown on 3,500 lx at 23°C for seven months.
Mycorrhizal observation and seedling growth assessment. X. affine NIFoS 2716 samples were washed and observed under a microscope (Leica DES8 APO; Leica Microsystems, Wetzlar, Germany). Mycorrhizae were placed in water and observed at 10× magnification. Hartig net samples of X. affine NIFoS 2716 were cut into 10 µm sections using a freezing microtome (Leica CM 1900; Leica Microsystems) and observed under a microscope (Leica DEDM 2500; Leica Microsystems). Growth characteristics of P. densiflora seedlings, including shoot height, and dry biomass of shoots and roots, were measured.
Statistical analysis
One-way analysis of variance (ANOVA) followed by Duncan’s test (p < 0.05) was performed to analyze the data. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA).
Results and Discussion
Mycelial growth of fungal species using different media and inorganic nitrogen sources
Mycelial growth on solid media. For in vitro mycorrhization, mycelia in liquid media were needed. Prior to liquid culture, we determined the optimal culture conditions for each species using solid media. Mycelial growth was evaluated on PDA, MEA, SDA, and MMN.
A. spissacea NIFoS 2719 showed better mycelial growth on SDA than in other media (Fig. 1). On the other hand, P. arhizus NIFoS 2784 showed optimum growth on PDA in 85 mm PDA petri dishes. However, P. arhizus NIFoS 2784 showed the least growth in MEA. S. spraguei NIFoS 2848 mycelia grew to less than 50 mm after 60 days of cultivation on all culture media tested and showed the least growth among all species examined. In addition, S. spraguei NIFoS 2848 showed no growth on SDA. X. affine NIFoS 2716 showed the fastest growth on MMN compared to the other species. X. affine NIFoS 2716 showed the highest growth after 30 days of inoculation on 85 mm petri dishes containing MMN. Both P. arhizus NIFoS 2784 and X. affine NIFoS 2716 showed the least growth on MEA. All four species were capable of growth on PDA, MEA, and MMN, although their preferred growth media were different. These results indicated that the four species can be cultivated on PDA and MMN.
Morphological analysis showed that the mycelial density of Amanita spissacea NIFoS 2719 was higher on PDA than on MEA and MMN (Fig. 2A~2D). P. arhizus NIFoS 2784 showed lower mycelial density on MMN than on PDA (Fig. 2E~2H). S. spraguei NIFoS 2848 on MMN exhibited low mycelial density with little aggregation (Fig. 2L). X. affine NIFoS 2716 showed lower mycelial density when cultivated on MMN than on PDA (Fig. 2M, 2P). Morphological analysis of mycelia in the different culture media revealed that all four species generally produce low mycelial density on MMN.
Mycelial growth on liquid media. All liquid media were based on MMN because ectomycorrhizal fungi are typically grown on MMN at pH conditions ranging from 5.8 to 6.2. Moreover, all four species showed low mycelial densities, which are ideal for liquid inoculation [6, 7]. We then measured the dry weights of the four species after cultivation in MMN media using different inorganic nitrogen sources. Inorganic nitrogen is abundant in forest ecosystems and is a major nutrient of ectomycorrhizal fungi [8]. Ammonium nitrogen is especially known to play an important role in promoting ectomycorrhizal growth [9].
A. spissacea NIFoS 2719 showed no differences in mycelial growth under different nitrogen sources (Fig. 3). P. arhizus NIFoS 2784 showed better mycelial growth in MMN and M2 than in M1 and M0. S. spraguei NIFoS 2848 produced abundant mycelia in MMN and showed the least growth in M0. Thus, our results were similar to those reported in a previous study [10]. Dry weights were approximately 2.9-fold higher in mycelia grown in MMN than those grown in M0. Mycelial growth in M2 was lower than in M1 media. As shown in Fig. 3, X. affine NIFoS 2716 showed the highest dry weight after cultivation in MMN. Dry weights of mycelia grown in MMN were approximately 2.8-fold higher than those grown in M0. Results showed small differences in mycelial growth after cultivation in different inorganic nitrogen sources.
Generally, ectomycorrhizal growth in MMN was higher than growth on inorganic nitrogen media [11], thereby indicating that inorganic nitrogen did not significantly enhance mycelial growth of the four fungal species. In addition, Jeon et al. [12] demonstrated that the highest dry weight of X. affine KFRI 1412 was obtained after cultivation on MMN medium.
Mycorrhizal formation
In this study, the four fungal species and Pinus densiflora were synthesized cultivated under sterile conditions. After seven months of inoculation, the roots of P. densiflora were observed. Only X. affine NIFoS 2716 showed mycorrhizal formation with P. densiflora (Table 1). The mycorrhization of Amanita spissacea has not been investigated. However, P. arhizus and S. spraguei have been previously shown to form mycorrhizae with pine hosts [13, 14]. S. spraguei formed ectomycorrhization with the Strobus (white pine), but showed little or no association with the Pinus. The P. densiflora species used in this is classified under the Pinus. Therefore, further studies must be conducted using other pine hosts.
Morphological analysis showed that the uninfected roots of P. densiflora had brownish lateral roots and abundant root hairs (Fig. 4A). Inoculated roots of P. densiflora showed simple and dichotomous lateral roots and rhizomorphs. Brown lateral roots were covered by dense mycelia, which were short and highly branched with numerous root tips and appeared white and light brown. The ends of the lateral roots were thicker than those of the uninfected roots. The hairs of inoculated roots were less developed than those of uninfected roots. The fungal mantles covered the root surface and formed the external mantle. Ectomycorrhizal symbiosis was identified based on the presence of the thick mantles of P. densiflora (Fig. 4D~4F).
Effect of X. affine inoculation on seedling growth
Conditions for cultivation of seedlings inoculated with X. affine NIFoS 2716 are important because inoculated seedlings in sterile conditions must adapt to the natural, non-sterile environment. In this study, we investigated shoot heights, dry weights of shoots and roots, and SR ratio (dry weight of shoot / dry weight of root) to evaluate seedling growth. The shoot heights of inoculated P. densiflora seedlings were significantly lower than those of the controls (Fig. 5A). On the other hand, the shoot weights of inoculated seedlings were higher, indicating that inoculation with promoted volume growth than elongation (Fig. 5B). The root dry weight of control and inoculated seedlings were 6.5 mg and 8.67 mg, respectively, which were lower than the obtained shoot dry weights (Fig. 5C). The dry weights of shoots and roots of the inoculated seedlings were approximately 1.8- and 1.3-fold higher than those of the controls, respectively. Seedlings inoculated with X. affine NIFoS 2716 showed significantly higher SR ratios than those of the controls (Fig. 5D). Root biomass was influenced by soil conditions; in particular, reduced availability of water or nutrients leads to lower SR ratios [15]. Accordingly, X. affine NIFoS 2716 inoculation exerted a positive effect on dry weights of shoots and roots.
In conclusion, our findings demonstrate the first evidence of ectomycorrhizal symbiosis of X. affine NIFoS 2716 and P. densiflora in vitro. In our symbiosis culture system, the liquid inocula were based on MMN medium to promote the growth of mycelia and maintain low mycelial density. Results of the morphological study showed that the inoculated roots of P. densiflora produced simple and dichotomous lateral roots and dense mycelia with white to light brown color. In addition, inoculation with X. affine NIFoS 2716 enhanced root and shoot developments of P. densiflora. The above results suggested that X. affine NIFoS 2716 can promote plant growth.
